1. Field of the Invention
This invention primarily relates to a method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, the steps of which involve the use of a pair of primers that allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, and a peptide nucleic acid (PNA) that acts as a PCR clamp as well as a sensor probe. This invention also relates to a kit for use in determining the presence of nucleotide variation(s) in the target polynucleotide sequence, which comprises the pair of primers and the PNA.
2. Description of the Related Art
Somatic mutations are present in various proportions in numerous developmental pathologies. Many diseases such as hemophilia, Albright syndrome (MAS), Alzheimer's disease, Huntington's disease, Duchenne muscular dystrophy (DMD), cystic fibrosis, etc., and a number of tumor pathologies are characterized by nucleotide variation(s) in the sequences of particular genes. These mutations/nucleotide variations may lead to a specific pathology when numbers of cells expressing the same reach a critical level.
Somatic mutations have been reported to be useful markers for early detection of cancers (S. Srivastava et al. (2001), Clin. Cancer Res., 7:1118-1126; F. R. Hirsch et al. (2001), Clin. Cancer Res., 7:5-22). For example, the K-ras gene, which encodes a 21-kDa GTP-binding protein, controls the mechanisms of cell growth and differentiation (C. Y. Chen et al. (2004), Clin. Chem., 50:481-489). The K-ras mutation in codons 12 and 13 occurs in 80-90% of pancreatic cancer and 35-50% of colorectal cancer (K. Motojima et al (1991), Am. J. Gastroenterol., 86:1784-1788; C. P. Dieterle et al. (2004), Clin. Cancer Res., 10:641-650; P. Anker et al. (1997), Gastroenterology, 112:1114-1120).
The major problem of using somatic mutations as markers of malignancy is that the clinical samples, especially body fluids or stools, frequently contain a trace amount of mutant DNA(s) in a large excess of wild-type DNA. The excess of wild-type DNA can exhaust essential reagents during PCR, and tends to mask the mutant DNA's signal during detection assays. The general strategy used to date to overcome this problem is to employ suppression of the wild-type allele or enrichment of the mutant allele during PCR amplification, followed by using a detection procedure that provides a sufficient resolution to reveal the mutant DNA's signal.
Methods used to enrich mutant template level include allele-specific amplification (H. linuma et al. (2000), Int. J. Cancer, 89:337-344), restriction enzyme digestion of wild-type DNA (C. P. Dieterle et al., (2004), supra; D. R. Jacobson and N. E. Mills (1994), Oncogene, 9:553-563; S. Norheim Andersen et al. (1996), Br. J. Cancer, 74:99-108), and sequence-specific ligation (D. A. Nickerson et al. (1990), Proc. Natl. Acad. Sci. USA, 87:8923-8927). These methods usually require subsequent procedures to detect mutant DNA's signal, including: (i) distinguishing the conformational or length differences by gel electrophoresis (T. Nishikawa et al. (2002), Clin. Chim. Acta, 318:107-112; S. Toyooka et al. (2003), Oncol. Rep., 10:1455-1459; M. Imai et al. (1994), Cancer, 73:2727-2733) or denaturing high-performance liquid chromatography (S. L. Lilleberg et al. (2004), Ann. N Y Acad. Sci., 1022:250-256); (ii) detecting short sequences by mass spectrometry (M. E. Lleonart et al. (2004), Nucleic Acids Res., 32, e53; X. Sun et al. (2002). Nat. Biotechnol., 20-186-189); and (iii) detecting nucleotide sequence changes by melting curve analysis (M. Nakao et al. (2000), Leukemia, 14:312-315), endonuclease V reaction (H. Pincas et al. (2004), Nucleic Acids Res., 32, e148) or hybridization on a microarray chip (M. Maekawa et al. (2004), Clin. Chem., 50:1322-1327). However, most of these methods are inconvenient for use in clinical laboratories due to multiple manipulations that are time-consuming and cost-inefficient. Most importantly, these methods increase the risk of contamination during multiple transfers.
Recently, the peptide nucleic acid (PNA)-based PCR procedure has been developed for the enrichment of mutant alleles (D. B. Demers et al., (1995), Nucleic Acids Res., 23:3060-3055). PNA is a synthetic DNA analog in which the normal phosphodiester backbone is replaced with a N-(2-aminoethyl)glycine chain. Its nucleobases complement DNA or RNA in the normal A-T and G-C geometry (P. E. Nielsen et al. (1991), Science, 254: 1497-1500; J. C. Hanvey et al. (1992), Science, 258:1481-1485; M. Egholm et al. (1993), Nature, 365:566-568). With the artificial backbone, PNA is resistant to nuclease activities.
Two important features make PNA a superior PCR clamp for specific alleles. It cannot serve as a primer for polymerization, nor can it be a substrate for exonuclease activities of Taq polymerase. In addition, the melting temperature (Tm) of a perfectly matched PNA-DNA duplex is higher than that of a DNA-DNA duplex with the same length PNA-DNA duplex is more stable than DNA-DNA duplex. A single mismatch in the PNA-DNA hybrid will cause a Tm drop of 10-18° C., which is much higher than that of the DNA-DNA duplex (E. M. Kyger et al. (1998), Anal. Biochem., 260:142-148). Therefore, within an appropriate temperature range, PNA can specifically block primer annealing or chain elongation on a perfectly matched template without interfering with reactions on templates with mismatched base(s) (X. Sun et al. (2002), supra; C. Thiede et al. (1996), Nucleic Acids Res., 24:983-984; Taback, B. et al. (2004), Int. J. Cancer, 111:409-414), which is known as “PNA-mediated PCR clamping” (H. Orum et al., (1993), Nucleic Acids Res., 21:5332-5336). In addition, the large Tm difference between perfectly matched and mismatched hybrids makes PNA a good sensor of point mutations. For example, a PNA sensor probe has been used to detect GNAS mutations after PCR (A. Karadag et al. (2004), Nucleic Acids Res., 32, e63).
PNA-mediated PCR clamping has been widely used for enrichment of rare mutant polynucleotides, including mutations in K-ras gene (B. Taback et al. (2004), supra.) and in mitochondrial DNA (D. K. Hancock et al. (2002), Clin. Chem., 48-2155-2163), the uidA gene of Escherichia coli O157:H7 strain (T. Takiya et al. (2004), Biosci. Biotechnol. Biochem., 68:360-368), and the DNA polymerase gene of hepatitis B virus (T. Kirishima et al. (2002), J. Hepatol., 37:259-265; W. Ohishi et al. (2004), J. Med. Virol., 72:558-565).
US Patent Application Publication No. 2004/0014105A1 discloses methods for the selective enrichment of low-abundance polynucleotides in a sample. The method uses enzymatically non-extendable nucleobase oligomer (e.g., PNA) as a PCR clamp to selectively block polymerase activity on high abundance species in the sample, thereby resulting in an enrichment of less abundant species in the sample.
US Patent Application Publication No. 2004/0091905A1 discloses a method for detecting a mutant polynucleotide in a mixture of mutant polynucleotides, wild-type polynucleotides and unrelated polynucleotides. The method uses an extension primer complementary to a first target sequence in both the wild-type and mutant polynucleotides. The method further uses a blocking probe (e.g., PNA probe) complementary to a second target sequence in the wild-type polynucleotides but not in the mutant polynucleotides. Extension of the primers annealed to the first target sequence in mutant polynucleotides produces long extension products. Extension of the primers annealed to the first target sequence in wild-type polynucleotides is blocked by the blocking probe annealed to the second target sequence. Short extension products or no extension products are produced. The extension products are isolated and used in a polymerase chain reaction (PCR). The PCR preferentially amplifies the long extension products.
The use of melting curve analysis in combination with hybridization probe system provides a powerful tool for the detection of single base alterations. The hybridization probe system is most widely used for this purpose. This system usually comprises a pair of oligonucleotides, i.e., the anchor and the sensor, each labeled with a different fluorescent dye, such that fluorescence energy transfer occurs between the two when they anneal adjacent sites of a complementary PCR strand (P. S. Bernard et al. (1998), Am. J. Pathol., 153:1055-1061). The melting curve profile of the sensor probe that is designed to anneal to the variable region of a target gene allows for homogeneous genotyping in a closed tube (P. S. Bernard et al. (1998), supra).
Recently, C. Y. Chen et al. developed a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis (C. Y. Chen et al. (2004), Clin. Chem., 50:481-489). J. Däbritz et al. combined the PCR-clamping approach with melting curve analysis using mutant specific hybridization probes and wild-type specific peptide nucleic acids (PNAs) to determine the genotypes of the most frequent point mutation in codon 12 of the proto-oncogene Ki-ras in tissue and plasma samples of patients with pancreatic cancer (J. Däbritz et al. (2005), Br. J. Cancer, 92:405-412). In addition, hybridization probes have been combined with PNA-mediated PCR clamping for detection of variant bcr-abl allele in leukemia (K. A. Kreuzer et al. (2003), Ann. Hematol., 82:284-289). Their studies demonstrated that use of a PNA clamp in combination with a pair of hybridization probes in PCR allows for a homogeneous detection of rare mutant DNA in a closed tube. However, in their designs, the added PNA competed for DNA binding with the sensor probe. The sensor probe therefore should be mutation-specific, i.e., it complements one of the mutant alleles instead of the wild-type allele. This leads to a disadvantage that the mutation-specific probe limits the types of mutations that may be detected. As a consequence, for a selected target gene, a variety of probes need to be synthesized if more than one type of mutations are expected to occur in the selected target gene. These probes have to be tested for their efficiency and compatibility when used together in the same reaction.
In a previous study, the applicants developed a simple method to detect trace amounts of K-ras mutants by using PNA as both PCR clamp and hybridization probe in a capillary PCR reaction, in which a 17-mer PNA complementary to wild type sequence and spanning the codons 12 and 13 of K-ras oncogene was used to clamp PCR for the wild type allele but not mutant alleles. Moreover, the PNA was labeled with a fluorescent dye and used as a sensor probe. The mutant PCR products, with a mismatch to the PNA probe, have a Tm about 10° C. lower than that of the wild-type product (Chiuan-Chian Chiou and Ji-Dung Luo, Detection of Trace Amounts of Mutant K-ras DNA by Peptide Nucleic Acid as Both PCR Clamp and Sensor Probe, Poster Session Abstracts of The San Diego Conference Cool Tools and Hot Applications Nov. 18-20, 2004 San Francisco, Calif.).
While this method allows the amplification of trace mutant polynucleotide(s) in the existence of 1,000-fold wild-type polynucleotides, its sensitivity and reproducibility is poor. Therefore, the applicants endeavored to develop an improved method for detecting mutant polynucleotide(s) in clinical samples.
In the present invention, the applicants surprisingly found that the extension temperature and the position of PCR primers have great influences on the efficiency of PNA-mediated PCR clamping. Based on these new findings, it is possible to develop a method with high sensitivity for detecting trace mutant polynucleotide(s). The newly devised method also proves to have excellent reproducibility and, hence, can serve as a useful tool for detecting nucleotide variation(s) in a variety of organisms and for screening rare mutation(s) in many diseases such as cancers.